L'analyse protéomique du tissu cérébral pathologique dans la maladie de Parkinson (MP) est un enjeu majeur à l'identification des causes moléculaires de la dégénérescence en vue de développer des thérapies curatives. Jusqu'à présent, les études chez l'humain se sont limitées à l'analyse du cerveau post-mortem, trop souvent représentatif d'un stade très avancé de la maladie et potentiellement altéré par différents facteurs. Dans ce travail, nous avons exploité l'accès temporaire au cerveau parkinsonien durant l'implantation d'électrodes de stimulation, pour obtenir une information moléculaire du tissu cérébral, in vivo et à un stade moins avancé de la maladie. Afin d'optimiser notre stratégie, nous avons ensuite développé un outil dédié à la capture tissulaire dont l'efficacité et le caractère non lésionnel ont été validés in vivo chez le primate. Ce travail permet d'envisager l'analyse protéomique du cerveau parkinsonien « vivant » afin d'identifier les causes moléculaires de la MP. En revanche, cette approche tissulaire n'est pas envisageable pour un diagnostic en routine clinique. Aussi, de nombreux groupes s'intéressent à l'analyse protéomique du LCR en vue d'identifier des marqueurs diagnostiques. Dans cette optique, nous avons mis au point une stratégie, basée sur l'utilisation de nanoparticules (NPs) fonctionnalisées qui a permis un enrichissement considérable des profils protéiques observés en spectrométrie de masse. La reproductibilité et la possibilité d'automatiser intégralement la préparation des échantillons font de notre approche une solution adaptée à la recherche de marqueurs moléculaires diagnostiques de la MP dans le LCR. Nous avons aussi démontré l'intérêt de notre approche pour l'analyse protéomique du plasma et du globule rouge. Enfin, nous avons évalué la possibilité d'utiliser ces NPs in vivo, pour une capture des protéines directement dans la circulation sanguine. / Proteomics analysis of pathological brain tissue in Parkinson's disease (PD) is of great importance to understand the molecular aetiology of degeneration and to develop curative treatments. To date, published studies have been restricted to the analysis of human post-mortem tissue samples, frequently derived from advanced disease stage and potentially altered by several factors. In this project we took advantage of the temporary access to PD patient's brain during electrode implantation to obtain in vivo molecular information from cerebral tissue at earlier stage of the disease. We further developed a dedicated tool to improve our tissue harvesting approach, and validated its efficiency and non-lesion effects in vivo in monkeys. This work opens the way to the proteomic analysis of fresh human brain samples to elucidate molecular causes of degeneration in PD. However such tissue investigation approach remains invasive and cannot be used in routine clinical screening for PD diagnosis. Proteomics analysis of cerebrospinal fluid (CSF) constitutes a promising alternative to identify neuropathological diagnosis markers. For this purpose, we developed a nanoparticle-assisted strategy enabling the enrichment of CSF proteins detection by mass spectrometry. Reproducibility and high throughput potentiality of our approach demonstrate its compatibility with clinical proteomics for PD diagnosis biomarker research in CSF. We also demonstrated the interest of this NP strategy for plasma and red blood cells proteome analysis. Finally, we evaluated the ability to use these NPs for in vivo protein harvesting in blood.

Jusqu’à 40% des protéines sont connues pour fixer des métaux, ces hétéroatomes jouant un rôle capital dans la régulation, la catalyse ou le maintien de la structure de ces protéines. Ces métalloprotéines sont ubiquitaires et d’une importance primordiale dans les trois domaines du vivant. Cependant, les méthodes actuelles dédiées à l’identification des membres de cette grande famille dans les protéomes bactériens sont soit inadaptées pour des approches à grande échelle, soit présentent des performances relativement limitées en l’absence d’une structure tridimensionnelle résolue. Dans ce contexte, différents outils d’analyse de séquence ont été testés, en recherchant des descripteurs de ces protéines (e.g. motifs, domaines conservés, empreintes phylogénétiques). Pour pallier le relatif manque de sensibilité de ceux-ci, de nouveaux descripteurs ont été construits, dédiés spécifiquement à l’identification des protéines à centre fer-soufre : (i) des profils de co-conservation des ligands du métal et (ii) des profile-HMMs adaptés à la détection d’homologues distants. Les pouvoirs prédictifs respectifs de ces catégories de descripteurs ont été évalués sur un jeu de protéines fer-soufre expertisé, en les considérant soit séparément soit en combinaison. L’ensemble de ces descripteurs a finalement été intégré dans un modèle linéaire généralisé en utilisant la technique d’elastic-net. Le modèle prédictif obtenu a été évalué sur le protéome complet d’Escherichia coli, sur lequel il atteint une précision de 89% et une sensibilité de 83%. Enfin, il a été appliqué à environ 300 protéomes pour explorer différentes relations biologiques comme l’abondance relative des protéines Fe-S et la tolérance à l’oxygène des organismes auxquelles elles appartiennent. / Up to 40% of all proteins are known to bind metals, the intrinsic metal atoms providing catalytic, regulatory and/or structural roles critical to their functions. These metalloproteins are ubiquitous and of major importance within the three domains of life. However, current methods dedicated to identifying members of this large family within bacterial proteomes are either not suitable for large-scale approach or are of relatively limited performance when no 3D structural template is available. Within this context, different sequence analysis tools relying on different category of protein descriptors (e.g. patterns, conserved domains, phylogenetic prints) were assessed. To overcome their relative lack of sensibility, new descriptors, specific towards iron-sulfur proteins identification were built: (i) co-conservation profiles of the metal ligands and (ii) tailored profile-HMMs for remote homologs detection. Their respective predictive power towards the identification of a manually curated iron-sulfur proteins dataset were assessed, either separately or in combination. All relevant descriptors were finally gathered into a generalized linear model by using the elastic-net method. The predictive model has been evaluated on Escherichia coli whole proteome resulting in a precision of 89% and a recall of 83%. Eventually, it has been applied to 300 proteomes allowing investigating different biological relationships, such as iron-sulfur proteins relative abundances and the oxygen dependency of bacterial organisms.

Orientador: Rogério de Castilho Jacinto / Banca: João Eduardo Gomes Filho / Banca: Paulo Carvalho Tobias Duarte / Resumo: Este estudo teve como objetivo comparar quantitativamente a diferença de expressão proteica na progressão da patogênese pulpar, bem como correlacionar as funções biológicas das proteínas identificadas no tecido pulpar normal, inflamado ou necrótico. As amostras foram obtidas de pacientes atendidos na Clínica Endodôntica da Faculdade de Odontologia de Araçatuba para tratamento endodôntico, sendo divididos em três grupos: grupo de polpa normal, com amostras do tecido pulpar obtidas a partir de dentes extraídos por indicação ortodôntica (n = 2); grupo de polpa inflamada, com amostras obtidas de pacientes com diagnóstico de pulpite irreversível (n = 2) e grupo de polpa necrótica, cujas amostras foram obtidas de pacientes com diagnóstico de periodontite apical crônica (n = 2). Após o preparo proteômico prévio, as amostras de polpa dentária foram processadas para análise proteômica quantitativa livre de marcadores em um sistema nanoACQUITY UPLC-Xevo QTof MS. A diferença na expressão entre os grupos de polpa normal e inflamada e grupos de polpa inflamada e necrótica foi calculada com o software Protein Lynx Global Service, usando o algoritmo Monte-Carlo, e expressa como p <0,05 para proteínas presentes em menor abundância e 1-p> 0,95 para proteínas presentes em maior abundância. Um total de 465 proteínas humanas foram identificadas em todos os grupos. Nos grupos normal, inflamado e necrótico, foram encontradas 241, 240 e 124 proteínas, respectivamente. Na análise quantitativa, as pr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to quantitatively compare the difference in protein expression in the progression of pulp pathogenesis, as well as to correlate the biological functions of proteins identified in normal, inflamed or necrotic pulp tissue. The samples were obtained from patients treated at the Endodontic Clinic of the Araçatuba Dental School for endodontic treatment, and were divided into three groups: normal pulp group with pulp tissue samples obtained from orthodontic teeth (n = 2) ; inflamed pulp group, whose samples were obtained from patients diagnosed with irreversible pulpitis (n = 2) and necrotic pulp group, whose samples were obtained from patients diagnosed with chronic apical periodontitis (n = 2). After previous proteomic preparation, dental pulp samples were processed for label-free quantitative proteomic analysis in a nanoACQUITY UPLC-Xevo QTof MS system. The difference in expression between the normal and inflamed pulp groups and groups of inflamed and necrotic pulp was calculated using the Protein Lynx Global Service software using the Monte Carlo algorithm and expressed as p <0.05 for proteins present in lower abundance and 1-p> 0.95 for proteins present in greater abundance. A total of 465 human proteins were identified in all groups. In the normal, inflamed and necrotic groups, 241, 240 and 124 proteins were found, respectively. In the quantitative analysis, the most expressed proteins were hemoglobin, peroxiredoxins and immunoglobulins, whereas the less expressed were the tubulins in the inflamed pulp group in relation to the normal pulp group. Expression of albumins, immunoglobulins and alpha-2-macroglobulin were increased in the necrotic pulp group when compared to normal pulp, whereas hemoglobin and actin were less expressed. As for the qualitative analysis, the proteins identified in the normal... (Complete abstract electronic access below) / Mestre

Le mélanome cutané est le plus agressif des cancers de la peau. Une fois métastasé, les options thérapeutiques sont limitées et peu efficaces. Dans le but de trouver de nouveaux marqueurs d'agressivité du mélanome cutané, une étude protéomique comparative entre deux lignées de mélanome murin, génétiquement semblables mais avec des agressivités différentes, a permis d'identifier l'annexine A1 (ANXA1) comme une protéine pro-invasive. Le but de ce travail de thèse a été d'évaluer la valeur pronostique d'ANXA1 et de déterminer son rôle et son mode d'action dans les processus d'invasion des mélanomes humains. Lors d'une étude immunohistologique rétrospective sur deux centres (Clermont-Ferrand et Saint-Etienne), il a été observé, indépendamment de l'indice de Breslow, une corrélation inverse entre le taux d'ANXA1 dans les tumeurs primitives de 61 patients et le délai d’apparition des métastases. Cette association est due à l’implication d’ANXA1 dans les processus d’invasion. En effet,nous avons démontré dans différentes lignées de mélanome qu'ANXA1 extracellulaire stimule les récepteurs aux peptides formylés (FPRs) exprimés par ces cellules. Cette fixation aux FPRs induit les voies des MAPK et STAT3 qui entraînent l'activation des métalloprotéases (MMP2). L'induction des MMP2 par ANXA1 augmente significativement le pouvoir invasif des lignées de mélanome. Nous avons aussi démontré qu’ANXA1 est transloquée coté externe de la membrane cytoplasmique là où elle subit un clivage par une sérine protéase qui pourrait être la nicaline. Ce clivage qui se produit après la sérine 28 n'a pas été décrit et jouerait un rôle dans la capacité invasive des mélanomes en libérant un ou des peptide(s) proinvasif(s). A long terme, ce travail vise à utiliser ANXA1 comme marqueur pronostique et/ou cible thérapeutique du mélanome cutané. / Cutaneous melanoma is the most aggressive skin cancer. Treatment options are limited and inefficient when melanoma has metastasized. In order to identify new markers of melanoma dissemination, protein profiles of two genetically similar murine melanoma cell lines have been compared. Both B16F10 and B16Bl6 cells induced primary tumours after subcutaneous graft, however, only B16Bl6 melanomas develop metastases. Among proteins differentially expressed, annexin A1 (ANXA1) is overexpressed in the aggressive B16Bl6 melanoma.The aim of the present work was to assess ANXA1 prognostic value in human melanoma and todecipher its implication in the invasion process. We report that, regardless of Breslow index, ANXA1 expression in primary tumours of 61 patients is inversely correlated with time to metastasis. This correlation is due to ANXA1 involvement in the invasion processes. Indeed, we show that in different human melanoma cell lines, extra cellular ANXA1 stimulates Formylated Peptide Receptors (FPRs). FPRs/ANXA1 interaction induces MMP2 activity through MAP Kinase and STAT3 pathways. ANXA1-stimulated MMP2 induces a significant increase of cell invasion ability. We also show that ANXA1 is externalized and localized on the cell surface where it is cleaved by a serine protease, which could be nicalin. ANXA1 cleavage occurs after Serine 28, a so far not described site. These data suggest that ANXA1 cleavage might be associated with invasion ability of melanoma cells by release of proinvasive peptide(s).These findings identify ANXA1 as a melanoma proinvasive protein that might be a promising prognosis marker and therapeutic target.

RESUME : Le Quorum Sensing est un système de communication bactérien permettant à une population de coordonner l’expression de gènes cibles en fonction de sa densité ou des propriétés du milieu (diffusion, flux....). Chez les bactéries à Gram négatif, le Quorum Sensing est basé sur la synthèse et la détection de petites molécules signal appelées N-acyl-homosérine lactones (AHLs). Les régulateurs transcriptionnels de type LuxR sont les médiateurs de ce système de régulation. Lorsque la concentration en AHLs augmente, ces molécules se fixent au domaine N-terminal d’un régulateur LuxR et provoquent des changement conformationnels entraînant une modification de l’activité du régulateur. Un tel système de régulation a été mis en évidence chez la bactérie Gram négative Brucella melitensis. Cette bactérie pathogène intracellulaire synthétise une dodécanoyl-homosérine lactone (C12-HSL) et possède deux régulateurs de type LuxR : VjbR et BabR. VjbR est impliqué dans la virulence de B. melitensis et est indispensable à l’expression de deux facteurs de virulence: le système flagellaire et le système de sécrétion de type IV VirB. Les C12-HSL ont quant à elles un effet répresseur sur ces deux structures membranaires. Durant ce travail, la mutation du domaine Nterminal du régulateur VjbR a permis de démontrer la capacité de VjbR à médier l’effet des C12-HSL sur l’opéron virB. Les souches mutées dans le gène vjbR forment des agrégats en cultures liquides. Nous avons montré que ce phénotype est lié à la production d’un exopolysaccharide, suggérant pour la première fois que Brucella pourrait former des structures de type biofilm. Cette étude a également permis de mettre en évidence un rôle majeur de VjbR dans la régulation de structures de surface puisque ce régulateur est impliqué dans le contrôle de l’expression de nombreuses protéines de membrane externe (Omp). L’utilisation de la technique d’immunoprécipitation chromatinienne (ChIP) a permis de montrer que VjbR régule directement deux de ces Omps ainsi que l’opéron virB. La virulence de Brucella est en partie basée sur sa capacité à dévier le trafic intracellulaire de ses cellules hôtes (phagocytes professionnels et nonprofessionnels) et à s’y multiplier. Lors de son cycle infectieux, Brucella est confrontée à de nombreux stress et environnements différents, suggérant la nécessité d’une régulation génétique fine en réponse à des stimuli environnementaux. Le Quorum Sensing, de par son implication dans la virulence de ce pathogène pourrait être impliqué dans de telles régulations. Afin d’aborder de façon globale le rôle de VjbR et de BabR chez B. melitensis, des études transcriptomique et protéomique des mutants ΔvjbR et ΔbabR ont été réalisées. Ces études ont permis de mettre en évidence que le Quorum Sensing chez B. melitensis est un système de régulation global, puisqu’il permet de réguler 10% du génome dans les conditions testées (dont 9% sous le contrôle de VjbR). De nombreuses cibles de ces régulateurs sont impliquées dans la virulence et l’adaptation aux conditions de stress (oxydatif, métabolique...), suggérant un rôle important du Quorum Sensing dans l’accomplissement du cycle infectieux de B. melitensis. SUMMARY : Quorum Sensing is a bacterial communication system wich allows the coordinated gene expression within a population regarding its density and environmental properties (diffusion, flow...). In Gram negative bacteria, Quorum Sensing is based on the synthesis and the detection of small diffusible molecules called N-acyl-homoserine lactones (AHLs). LuxR transcriptional regulators are the mediators of these regulation systems. When AHL concentration increases, these molecules bind to the N-terminal domain of a LuxR-type regulator and leads to conformational changes driving the modification of the regulator activity. A similar regulation system has been discovered in the Gram negative bacteria Brucella melitensis. This intracellular pathogen synthesizes a dodecanoylhomoserine lactone (C12-HSL) and possesses two LuxR-type regulators: VjbR and BabR. VjbR is involved in the virulence of this pathogen and is crucial for the expression of two virulence factors : the flagellar system and the type four secretion system VirB. C12-HSL have a repressor effect on these two membrane structures. During this work, mutation of the N-terminal domain of VjbR allowed us to demonstrate the ability of VjbR to mediate C12-HSL effect on the virB operon. vjbR mutated strains aggregate in liquid cultures. We have demonstrated that this phenotype is linked to the production of an exopolysaccharide, suggesting for the first time that Brucella could form biofilm-type structures. This study also demonstrates that VjbR has a major role in the regulation of surface structures because this regulator controls the expression of many outer membrane proteins (Omp). Using the chromatin immunoprecipitation technique (ChIP), we have shown that two of these Omps, as well as the virB operon, are directly regulated by VjbR. The virulence of Brucella is partly based on its ability to deviate the intracellular traffic of its host cells (professional and nonprofessional phagocytes) and to proliferate within these cells. During its infectious cycle, Brucella faces numerous stresses and environments, suggesting the necessity of a finely tuned genetic regulation depending on environmental stimuli. Quorum Sensing, through its involvement in the virulence of this intracellular pathogen, could be involved in such regulations. In order to investigate the role of VjbR and BabR in B. melitensis, global transcriptomic and proteomic studies of ΔvjbR and ΔbabR mutants were performed. These studies demonstrate that Quorum Sensing is a global regulation system in B. melitensis because it controls the expression of 10% of the genome in the condition tested (9% through VjbR). Numerous targets of these two regulators are involved in virulence and adaptation to environmental stresses (oxydative, metabolic...), suggesting an important role of Quorum sensing in the achievement of the infectious cycle of B. melitensis.

Controlled Exposure Experiments revealed lesions in the statocysts of four cephalopod species of the Mediterranean Sea (Sepia officinalis, Loligo vulgaris, Illex coindetii and Octopus vulgaris), when exposed to relatively low intensity low frequency sounds. The analysis was performed through: scanning (SEM) and transmission (TEM) electron microscopy techniques of the whole inner structure of the cephalopod statocysts, especially on macula and crista; SEM of the epidermal lines of cephalopod hatchlings; and proteomic studies (2DE/MALDI –MS) of the statocyst’s endolymph. All exposed adult individuals presented the same lesions and the same incremental effects over time, consistent with a massive acoustic trauma observed in land species that were exposed to much higher intensities of sound. Immediately after exposure, the damage was observed in the macula statica princeps (msp) and in the crista sensory epithelium. Kinocilia on hair cells were either missing or were bent or flaccid. A number of hair cells showed protruding apical poles and ruptured lateral plasma membranes, most probably resulting from the extrusion of cytoplasmic material. Hair cells were also partially ejected from the sensory epithelium, and spherical holes corresponding to missing hair cells were visible in the epithelium. The cytoplasmic content of the damaged hair cells showed obvious changes, including the presence of numerous vacuoles and electron dense inclusions not seen in the control animals. The appearance of these lesions became gradually more pronounced in individuals after 12, 24, 48, 72, and 96 hours. Special attention was given to validate these findings with control animals that were caught, maintained and sequentially sacrificed following the same protocol as the exposed individuals. The statocyst ultrastructure was therefore revisited and a comparative analysis was carefully conducted to assess the lesions triggered by the exposure to noise This study also presents preliminary results of the sound effects on epidermal lines of cephalopod hatchlings. The lesions, consistent with an acoustic trauma, were identic in the three species that were exposed, but their evolution over time, in opposition with what was observed in the statocysts, were different, suggesting that the animal size and metabolic response might play a role in a possible recovery process. The analysis of noise effects in the statocyst endolymph by proteomic techniques was only conducted on Sepia officinalis. The presence of differential staining of gels from control and subjected to sound exposure individuals demonstrate that the injuries could be related to a possible physiological imbalance that would affect the protein levels of the endolymph. The lesions and findings described here are new to cephalopod pathology. Given that lowfrequency noise levels in the ocean are increasing (e.g. due to shipping, offshore industry, and naval maneuvers), that the role of cephalopods in marine ecosystems is only now beginning to be understood, and that reliable bioacoustic data on invertebrates are scarce, the present study and future investigations will bring an important contribution to the sustainable use of the marine environment. / Després de sotmetre'ls a experiments d'exposició controlada a sons de baixa intensitat i baixa freqüència es van observar lesions en els estatocists de quatre espècies de cefalòpodes de la mar Mediterrània (Sepia officinalis, Loligo vulgaris, Illex coindetii i Octopus vulgaris). L'anàlisi es va realitzar per mitjà de de microscòpia electronica d'escombratge (SEM) i de transmissió (TEM) de tota l'estructura interna de l'estatocist dels cefalòpodes, especialment en la màcula i en la crista, per SEM de les línees epidèrmiques de les larves dels cefalòpodes i per tècniques de proteòmica (2DE/MALDI-MS), de l'endolimfa de l'estatocist. Tots els estatocists d'individus adults de cefalòpodes exposats presentaven les mateixes lesions i aquests efectes eren més greus a mesura que passava el temps després de l'exposició als sons. Tots els animals exposats al soroll van mostrar lesions consistens amb trauma acústic massiu observat en altres espècies terrestres que havien estat exposades a intensitats molt més altes de so. Immediatament després de l'exposició, es van observar danys a la macula statica princeps (msp) i en l'epiteli sensorial de la crista. Els quinocilis de les cèl·lules ciliades desapareixien o es doblegaven i es tornaven flàccids. Un nombre important de cèl·lules ciliades mostraven els pols apicals sobresortint de l'epiteli sensorial, així com el trencament de les membranes plasmàtiques laterals, molt probablement com a resultat de l'extrusió de material citoplasmàtic. Les cèl·lules ciliades també van ser parcialment expulsades de l'epiteli sensorial deixant visibles forats esfèrics en el mateix. El contingut citoplasmàtic de les cèl·lules ciliades danyades va mostrar canvis obvis, com ara la presència de nombrosos vacúols i inclusions electrodenses que no es veien en els animals control. L'aparició d'aquestes lesions es va tornar gradualment més pronunciada en els individus analitzats després de 12, 24, 48, 72 i 96 hores. Es van validar curosament aquests resultats per mitjà de la comparació amb els animals control que van ser capturats, mantinguts i sacrificats de forma seqüencial seguint el mateix protocol que els individus exposats. La ultraestructura de l'estatocist va ser revisada i es va dur a terme un curós anàlisi comparatiu per tal d'avaluar les lesions provocades per l'exposició al soroll. Aquest estudi també presenta els resultats preliminars dels efectes del so en les línies epidèrmiques de cefalòpodes recent nascuts. Les lesions, consistens amb trauma acústic, eren idèntiques en les tres espècies que van ser exposades, però la seva evolució en el temps, en oposició amb el que es va observar en els estatocists, era diferent, cosa que suggereix que la grandària dels animals i la resposta metabòlica podria tenir influència en un possible procés de recuperació. L'avaluació dels efectes en l'endolimfa de l'estatocist per tècniques de proteòmica es va dur a terme només en Sepia officinalis. La presència de taques diferencials en els gels dels individus control i els sotmesos a exposició a so demostren que les lesions podrien estar relacionades amb un possible desequilibri fisiològic que tindria repercusions en els nivells proteics de l'endolimfa. Les lesions descrites aquí són noves pel que fa a la patologia dels cefalòpodes. Atès que els nivells de soroll de baixa freqüència a l'oceà estan augmentant (per exemple, a causa del transport, la indústria petrolera i les maniobres navals), que el paper dels cefalòpodes en els ecosistemes marins només ha començat a ser entès recentment, i que les dades bioacústiques fiables sobre els invertebrats són escasses, el present estudi i les investigacions futures aportaran una important contribució a l'ús sostenible del medi marí.

Hepatocyte growth factor (HGF) and its specific receptor MET play a role in many physiological functions including proliferation, migration and morphogenesis. Recently, research results in our laboratory showed that recombinant HGF variants (NK1, NK2, NK3 and NK4) became antagonists to HGF/MET pathway by suppressing proliferation, migration and invasion in human breast cancer cells (MDA-MB-435S, MDA). Similar results were achieved when HGF variants genes were introduced in MDA cells. To understand the molecular mechanism of breast cancer cells metastasis suppressed by HGF variants, MDA and five transfectants, including MDA-GFP, MDA-NK1, MDA-NK2, MDA-NK3 and MDA-NK4 cells were used for proteomic analysis using two-dimensional electrophoresis (2-DE). Differential analysis revealed that a total of 56 polypeptides were differentially expressed through five sets of comparison using wild-type MDA cells as a control. A total of 17 polypeptides were shown differential expression between MDA and MDA-GFP cells, with 11 down-regulated and 6 up-regulated. Eighteen polypeptides were differentially expressed between MDA and MDA-NK1 cells, with 15 down-regulated and 3 up-regulated. There were 22 differentially expressed polypeptides found between MDA and MDA-NK2 cells, in which 14 were down-regulated and 8 were up-regulated. Sixteen polypeptides were shown differentially expressed between MDA and MDA-NK3 cells, with 11 down-regulated and 5 up-regulated. A total of 18 polypeptides were shown differential expression between MDA and MDA-NK4 cells, with 15 down-regulated and 3 up-regulated. Proteomic analysis showed that a total of 43 polypeptides were differentially expressed through four sets of comparison (MDA-GFP and MDA-NK1, MDA-GFP and MDA-NK2, MDA-GFP and MDA-NK3, and MDA-GFP and MDA-NK4). To understand the differential expression among different HGF variants-transfected MDA cells, three sets of cross analysis were also carried out (MDA-NK1 and MDA-NK2, MDA-NK1 and MDA-NK3, and MDA-NK1 and MDA-NK4) and the results showed that a total of 37 differentially expressed polypeptides were found in the three sets of comparison. Similarly, when MDA-NK2 cells were used as a control to compare with MDA-NK3 and MDA-NK4 cells, 34 significantly differential expressed polypeptides were found. The last set of comparison between MDA-NK3 and MDA-NK4 cells, 19 polypeptides were found significantly differential expression. Therefore, our current results revealed that the differentially expressed polypeptides in MDA-MB-435S cells and HGF variants-transfected MDA cells could be related to the inhibition of proliferation and migration of human breast cancer cells by HGF variants.

There is a thermal range for the operation of neural circuits beyond which nervous system function is compromised. Poikilotherms are particularly vulnerable to thermal stress, since their body temperature can fluctuate with ambient temperature. Animals that experience frequent hyperthermia have various coping mechanisms such as the thermoprotective effect of a prior exposure to sublethal temperatures (heat shock response). The molecular mechanisms of this thermoprotection have yet to be understood. This project studies the changes in protein expression in the nervous system of gregarious Locusta migratoria subjected to heat shock. For this purpose, proteins were extracted from metathoracic ganglia (MTG) by different methods and a proteomic map was subsequently obtained by 2-D gel electrophoresis which was compared between control (CON) and heat-shocked (HS) animals. Additionally, the localization pattern of Hsp70 was studied in the MTG of CON and HS gregarious locusts. Although 2-D gels showed changes in the amount of different isoforms of ATP-synthase β, the overall amount of this protein subunit was found to be unchanged. My experiments also revealed no significant change in the distribution of Hsp70 in the MTG of locusts caused by HS. However, new findings show that this protein is constitutively expressed at higher levels in perineurium, glia and tracheal cells than in neurons. In separate experiments, isolated locusts were also examined in order to measure any stress-associated increase of Hsp70 in the tissues of animals not previously exposed to crowding pressure. Quantitative western blots did not show a consistent change of the Hsp70 level in the MTG of isolated locusts following heat shock. Results of my research suggest that the change in the protein profile of the metathoracic ganglion following heat shock, if it exists, is subtle or occurs in very low-abundance proteins whose monitoring requires the application of special techniques. Alternatively, the thermoprotective effect of heat shock on the nervous system might be promoted through other pathways which can change the protein activity at the post-translational level and may work independently from protein synthesis. / Thesis (Master, Biology) -- Queen's University, 2009-03-20 12:28:32.962

In situ protein detection in human tissues using antibodies reveals the cellular protein localization, and affinity-based proteomic studies can help to discover proteins involved in the development of diseases. However, antibodies often suffer from cross-reactivity, and the lack of positive and negative tissue controls for uncharacterized proteins complicates the mapping of the proteome. The aim of this thesis is thus to improve the methodology for validating antibodies used for immunostaining on formalin-fixed paraffin-embedded tissues. Two of the papers include comparisons between mRNA-expression and immunostaining of corresponding protein. In paper I, ISH and IHC staining patterns were compared on consecutive TMA-slides. The study of well-characterized genes showed that ISH could be used for validation of antibodies. ISH was further used for antibody evaluation, and could validate four out of nine antibodies showing potentially interesting staining patterns. In paper III, transcriptomic data generated by RNA-sequencing were used to identify tissue specific expression in lymphohematopoietic tissues. An increased expression in one or more of these tissues compared to other tissue types was seen for 693 genes, and these were further compared to the staining patterns of corresponding proteins in tissues. Antibody labeling is necessary for many immunoassays. In paper II, two techniques for antibody-biotinylation were compared, aiming to find a stringent labeling method for antibodies used for immunostaining on TMAs. The ZBPA-method, binding specifically to Fc-part of antibodies, was found to be superior to the Lightning Link-biotinylation kit targeting amine groups, since labeling of amine groups on stabilizing proteins in the antibody buffer causes unspecific staining. The localization of the estrogen receptor beta (ERβ) in human normal and cancer tissues was studied in paper IV. Thorough evaluation of 13 antibodies using positive and negative control cell lines showed that only one antibody, PPZ0506, is specific for ERβ in all three immunoassays used. Contradictory to previously published data, tissue profiling using PPZ0506 showed that ERβ is expressed in a limited number of normal and cancer tissues. In conclusion, the present investigations present tools for validation of antibodies used for large-scale studies of protein expression in tissues.

A complete proteome analysis of the chloroplast stroma, using 2D-PAGE, from spinach and Arabidopsis was performed. To improve the identification of proteins a computer program named SPECLUST was used. In SPECLUST, peak masses that are similar in many spots cluster together because they originate from the same protein with different locations on the gel. Within this program peaks in a cluster can be investigated in detail by peaks-in-common, and the unidentified masses that differ between spots in a cluster could be caused by protein modifications, which was analysed further by MS/MS. The thylakoid is an internal membrane system in the chloroplast where protein complexes involved in photosynthesis are housed. Enclosed in the thylakoid membrane is the chloroplast lumen, with a proteome estimated to contain 80-200 different proteins. Because the chloroplast lumen is close to the photosynthesis machinery in the plant, one can expect that the lumen proteome will change depending on if the plant is dark or light adapted. DIGE analysis of lumen proteins found that 15 lumen proteins show increased relative abundance in light-adapted plants. In addition co-expression analysis of lumen protein genes suggests that the lumen protein genes are uniformly transcriptionally regulated, not only by light but in a general manner. Plastocyanin is one of the proteins involved in the electron transfer in photosynthesis. Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis, where PETE2 is the more abundant isoform. Knockout mutants of each of the plastocyanin isoforms shows that a 90% reduction of plastocyanin levels affects rates of photosynthesis and growth only slightly. A corresponding over-expression of plastocyanin in each of the two knockout mutants results in essentially wild-type photosynthetic performance. Reduced plastocyanin levels make the plant sensitive to Cu stress and therefore plastocyanin plays a major role as a Cu sink. A by-product of photosynthesis is hydrogen peroxide, which may be harmful for the plant. The discovery that an abundant protein found in the chloroplast lumen, TL29, shared sequence homology to Ascorbate Peroxidase (APX) was therefore of interest. We have evidence that TL29 is not an APX protein; it lacks the heme-binding active site and shows no activity. TL29 is located in the grana region and is electrostaticaly attached to the thylakoid membrane. It has four isoforms, with different pIs, both in the native and denatured form. It has no interaction with ascorbate, when compared to raAPX1. TL29 has two cysteine residues and one of them seems to have redox-regulated function, proposing that it may interact with other proteins close to PSII.